In Vitro Sperm–Epididymosomes Interaction Immediately Before Fertilization Changes Sperm Fertility Potential

Abstract

Background: Sperm acquire fertility ability during epididymal maturation mainly in the epididymal caput and corpus, and once matured, are stored in the epididymal cauda. During storage, interactions with cauda epididymosomes (epEVs) may influence sperm fertility potential; however, the role of such interactions on sperm fertility remains elusive. Objectives: To investigate the effect of epEVs on sperm fertility potential using the bovine model. Materials and Methods: A pool of epEVs from cauda epididymal fluid of five bovine males was characterized regarding size, concentration, morphology, and specific markers. To determine the sperm–epEVs interaction protocol, green-labeled epEVs (PKH67) were incubated with post-thawed cauda epididymal sperm from three bovine males at ratios of 500, 1000, or 2000 epEVs/sperm, and for incubation periods of 1.5, 3, or 6 h. After that, Hoechst-stained sperm were analyzed by flow cytometer. Green fluorescence percentage and intensity of 5000 positive Hoechst events were considered as sperm–epEVs interacting. Controls were performed by incubating sperm with PKH67 in PBS. A total of 49 microRNAs (out of 380 investigated) were found detected in the epEVs and the top five were investigated in sperm by quantitative polymerase chain reaction (qPCR) following sperm–epEVs interaction. Subsequently, sperm that interacted with epEVs were used to produce embryos by in vitro fertilization. Results: epEVs displayed 114.20 ± 3.60 nm, 3.48 × 109 ± 1.84 × 108 particles/mL, a cup-shaped morphology, and positivity for ALIX, CD-81, and CD-63 markers. Following determination of the protocol (1000 epEVs/sperm for 3 h), two microRNAs (miR-935 and -421) previously detected in epEVs, were found in sperm. Finally, sperm incubated with epEVs resulted in higher (p = 0.04) blastocyst rates (epEVs: 38.9% ± 7.3%; 58/149; control: 26.6% ± 5.6%; 40/145). Discussion and Conclusion: Incubation of sperm with 1000 epEVs/sperm for 3 h promoted sperm–epEVs in vitro crosstalk that enhanced blastocyst rates. These findings suggest that epEVs modulate paternal contribution to development and provide valuable insights to promote a fast and dynamic control of male fertility.